FIGURE 5.

Proposed catalytic mechanism for PseG based on modeling of the UDP-sugar complex and activity analysis of substitution derivatives. Here, PseG employs a single displacement mechanism involving C–O bond cleavage via direct attack of the anomeric carbon by a hydroxide nucleophile. Specifically, the residue His¹⁷ performs a base-catalyzed attack of water by abstracting a proton. The activated water is anchored with its other proton hydrogen-bonded to the main-chain carbonyl of Ile¹³, and it is further stabilized by the partially positively charged edge of the aromatic ring of Tyr⁷⁸ (not shown). Several PseG residues participate in direct intermolecular interactions with the sugar moiety and stabilize the Michaelis complex, including Asn²⁵⁵ that appears as a determinant in selecting the twist-boat conformation already present in the free substrate. This twist-boat conformation of the sugar appears primed for catalysis, as it exposes the C-1 atom and favors attack by the His¹⁷-activated water, resulting in inversion of stereochemistry at C-1 and cleavage of the C-1–O_Gly bond. The liberated sugar product is expected to attain an equilibrium distribution of α and β anomers that interconvert nonenzymatically in solution. The inset shows a proposed scheme for His¹⁷ acting as a general base, abstracting a proton from water, or via the H17N substitution H-bonding with the proposed catalytic water molecule.